Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

doi: 10.1073/pnas.2020695118

Figure Lengend Snippet: IP6K2 binds to CK-B. (A) Protein sequence of CK-B of length of 381 amino acids. Nine CK-B peptides (highlighted in yellow) were identified from IP6K2 immunoprecipitates using LC-MS/MS. (B) Cellular localization of IP6K2 and CK-B in the cerebellum was determined by probing IP6K2 with Alexa-488–tagged secondary antibody and CK-B with Alexa-568 secondary antibody. The expression of the proteins was observed through a confocal microscope in the Purkinje and the granule cell layer in the cerebellum of WT mouse brain sections (40-μm thickness). (Scale bar, 50 μm.) (C) IP6K2 immunoprecipitates were blotted against CK-B in both neuronal cells and cerebellar brain lysates (lane 1: IP6K2 immunoprecipitate in N2A neuronal cells blotted against CK-B; lane 2: IgG control; lane 3: IP6K2 immunoprecipitate in cerebellar brain lysate blotted against CK-B). (D) IP6K isoforms were immunoprecipitated, and the blot was probed with CK-B antibody. The result of CK-B selectively binding to the IP6K2 isoform was also confirmed by LC-MS/MS. (E) Comparison of CK-B expression in the WT- and IP6K2-KO cerebellar lysates by Western blot. (F) Quantitation of CK-B expression. (G) Western blot depicts the expression of CK-M in WT and IP6K2-KO mice. (H) The activity of both forms of creatine kinase (B and M) was determined in WT and K2-KO mice in the brain and muscles, respectively. (I) CK-B immunoprecipitates from WT and K2-KO cerebella were immunoblotted with an anti-ubiquitin antibody. (J) CK-B binding to K222A kinase dead IP6K2 mutant to examine kinase dependence on binding. Data are representative of three independent experiments performed under identical conditions. Data are presented as the mean ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05 analyzed by two-tailed Student t test. CK-B peptides detected through LC/MS-MS with their respective peptide identification probability, mascot ion score, and mascot identity scores are listed in SI Appendix, Table S1.

Article Snippet: IP6K2 short hairpin RNA (shRNA), CK-B shRNA, and control shRNA plasmids were procured from Santa Cruz Biotechnology.

Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy, Expressing, Microscopy, Immunoprecipitation, Binding Assay, Western Blot, Quantitation Assay, Activity Assay, Mutagenesis, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

doi: 10.1073/pnas.2020695118

Figure Lengend Snippet: IP6K2 deletion elicits decreased adenylate pool as well as a decrease in the total brain ATP and phosphocreatine levels. (A) Chromatogram represents the adenine nucleotide peaks identified through HPLC using the cerebellar lysates of wild-type and IP6K2-KO mice. (B and C) The total adenylate pool (sum of AMP, ADP, and ATP) in the mice cerebella (B) as well as in N2A cells (C) with and without IP6K2. (D and E) ATP levels in cerebella from WT and IP6K2-KO mice (D) as well as WT and IP6K2 knocked-down N2A cells (E). (F) Comparison of ATP levels through quantitation of HPLC chromatogram in WT, IP6K2 knocked-down N2A cells, and N2A cells transfected with IP6K2. (G) Comparative HPLC profiles for detection of PCr levels in the WT and IP6K2-KO mouse brain, liver, and kidney, the three major organs where creatine/phosphocreatine is processed. (H) Quantitation of relative amount of phosphocreatine in WT and K2-KO mouse brain, liver, and kidney lysates. Data are representative of three independent experiments performed under identical conditions. Data are presented as the mean ± SD: ***P < 0.001, **P < 0.01, and *P < 0.05 analyzed by two-tailed Student t test.

Article Snippet: IP6K2 short hairpin RNA (shRNA), CK-B shRNA, and control shRNA plasmids were procured from Santa Cruz Biotechnology.

Techniques: Quantitation Assay, Transfection, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

doi: 10.1073/pnas.2020695118

Figure Lengend Snippet: IP6K2 deletion leads to decreased expression of mitochondrial complex III and reduced expression of cytochrome c1 subunit as well as impaired OXPHOS. (A) Scheme of mitochondrial electron transport chain complexes (I–V). (B) Expression of electron transport chain complexes (ETC) (CI–CV) using total OXPHOS mixture antibody was assessed in WT and K2-KO mouse cerebellum. The premixed antibody mixture contained five mouse monoclonal antibodies, one each against CI—NADH: ubiquinone oxidoreductase subunit B8 (NDUFB8), 20 kDa; CII—succinate dehydrogenase subunit-B (SDHB), 30 kDa; CIII—ubiquinol-cytochrome c reductase core protein 2 (UQCRC2), 48 kDa; CIV—mitochondrially encoded cytochrome c oxidase subunit I (MTCO1), 40 kDa; and CV—ATP synthase alpha-subunit (ATP5A), 55 kDa. (C) Quantitation of the expression of ETC complexes I–V in mitochondria from WT and K2-KO mouse cerebellum. (D) Expression of complex III subunits (cytochrome c1, cytochrome b) as well as total complex III using Western blotting. (E) Quantitation of the expression of these proteins depicted in a histogram. (F) N2A cells were transfected with IP6K2 shRNA (lane 2) and double-transfected using IP6K2 and CK-B shRNA (lane 3). The expression of cytochrome c1 and the total complex III was assessed in N2A cells using Western blotting. (G) The relative change in complex III activity in isolated mitochondria of WT and K2-KO mouse cerebellum was measured using a colorimetric mitochondrial complex III activity assay. (H) OCR. (I) Basal respiration, ATP production, maximal respiration, spare capacity, proton leak, and nonmitochondrial respiration were determined in WT, K2 knocked-down (K2-KD), and CK-B + K2 double-knocked-down (CK-B KD+K2-KD) N2A cells using Seahorse MitoStress Kit. Data are presented as the mean (n = 3) ± SD. ***P < 0.001, **P < 0.01, and *P < 0.05, analyzed by one-way ANOVA and two-tailed Student t test.

Article Snippet: IP6K2 short hairpin RNA (shRNA), CK-B shRNA, and control shRNA plasmids were procured from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitation Assay, Western Blot, Transfection, shRNA, Activity Assay, Isolation, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

doi: 10.1073/pnas.2020695118

Figure Lengend Snippet: IP6K2 deletion elicits increased oxidative stress and neuronal damage which is restored by treating the neurons with NAC and PCr. (A) Proteins oxidized by oxygen free radicals are modified by the addition of carbonyl groups into their side chains. The OxyBlot analysis determines these carbonyl groups as a hallmark of oxidation of proteins. Oxidative modification of proteins in the WT and K2-KO brain cerebellum was detected using this technique. (B) Relative mitochondrial reactive oxygen species were assessed in the isolated mitochondria from the WT and K2-KO brain cerebellum. (C) Golgi staining was performed to determine the Purkinje cell morphology in the cerebellar sections of WT and K2-KO mice (n = 5). The cell volume was quantified and calculated using Image J software. (D) Comparative anxiety-like behavior of WT and K2-KO mice was determined using the elevated plus maze test. Elevated plus maze test centers on the fear of open and elevated spaces to mice. Arranged in a plus shape, the elevated plus maze has two closed and two open arms, as well as an open center as illustrated in the scheme. The time spent as well as the distance traveled by WT and K2-KO mice is measured and is represented in the figure using a pie graph (n = 12). (E) Both WT and K2-KO mice were trained to perform the treadmill fatigue/exhaustion test. In this experiment, each mouse is allowed to run for about 10 min over a conveyor belt with gradually increasing speed as illustrated in the scheme of experimental design. The maximum attained running speed and the time of exhaustion were calculated for the experimental mice (n = 12) as represented in the histogram. (F) The primary neurons were treated with a mixture of mitochondria-specific antioxidant, NAC, and an ATP source, PCr, for 5 d. The branching of primary neurons was reversed to near WT levels with cotreatment of NAC and PCr which was determined by staining them against α-tubulin with Alexa-568–tagged secondary antibody. (G) The dendritic branching in primary neurons was quantified using the Sholl analysis in ImageJ software. (H–J) IP6K2 knocked-down N2A cells were treated with PCr and NAC to determine the relative levels of ATP, ROS, and cell viability between WT, K2-knocked-down cells, and treated cells. Data are presented as the mean (n = 3) ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, analyzed by two-tailed Student t test and one-way ANOVA.

Article Snippet: IP6K2 short hairpin RNA (shRNA), CK-B shRNA, and control shRNA plasmids were procured from Santa Cruz Biotechnology.

Techniques: Modification, Isolation, Staining, Software, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: IP6K2 binds 4.1N in the brain. A, Silver staining of SDS-PAGE of IP6K2 immunoprecipitates (lane 2) from wild-type mouse brain lysate showed the presence of 4.1N protein (130 kDa) as a potential interacting partner of IP6K2 against IgG (lane1) as a control. B, Seventeen (17) 4.1N peptides (highlighted in yellow) were identified from IP6K2 immunoprecipitates using LC-MS/MS to confirm the identity of the 4.1N protein. C, Coimmunoprecipitates of IP6K2 from WT (lane 2) and IP6K2-knockout (K2-KO; lane 3) mouse brain lysates immunoblotted (IB) with 4.1N antibody confirmed the binding to 4.1N protein against IgG immunoprecipitate (IP) as a control (lane 1). D, Coimmunoprecipitates of IP6K2 blotted against 4.1N in neuronal cells also revealed its association with the 4.1N protein. Lane 1, IgG immunoprecipitate; lane 2, IP6K2 immunoprecipitates from N2A cell lysates; lane 3, IP6K2 immunoprecipitates from PC-12 cell lysates. E, GST-DNA constructs of different fragments of IP6K2 were transfected into N2A cells. Coimmunoprecipitates of such IP6K2 constructs expressing cell lysates were blotted against 4.1N antibody. This revealed the binding site of IP6K2 with 4.1N to be present between residues 202 and 261 of IP6K2. F, Coimmunoprecipitation of IP6K2 from wild-type mouse brain lysates (lane 1) and IP6K2-KO mice brain lysates (lane 2, control) showed the binding of 4.1N and 4.1B to IP6K2. However, 4.1G and 4.1R isoforms of the 4.1 family of proteins did not bind to IP6K2. G, Coimmunoprecipitates of 4.1N from mouse brain lysates were blotted against all three isoforms of IP6K (K1, K2, and K3). Corresponding immunoblots against IgG immunoprecipitates as a control (lane 2) showed selective binding of the 4.1N protein to IP6K2 and not to the IP6K1 or IP6K3 isoforms. H, Full-length IP6K2 and IP6K2 kinase dead mutants (K222A) were transfected into N2A cells. Immunoblots of the lysates from both cells showed confirmatory binding of 4.1N (lane 1) when compared with their IgG controls (lane 2) indicating that the binding of 4.1N to IP6K2 is not dependent on the kinase activity of the latter. Data are representative of three independent experiments performed under identical conditions. 4.1N peptides detected through LC/MS-MS with their respective peptide identification probability, mascot ion score and mascot identity score, Table 1-1.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Silver Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Knock-Out, Binding Assay, Construct, Transfection, Expressing, Western Blot, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: Nuclear translocation of 4.1N requires IP6K2 facilitation. A, Western blots show endogenous expression of IP6K2 in the cytosolic and nuclear fractions of wild-type mouse brain lysates. B, Corresponding comparisons of 4.1N expression in the wild-type brain lysates and IP6K2-KO brain lysates. C, The relative levels of the protein bands in the immunoblot depicted in A and B estimated through normalization against their relevant inputs are shown in the histogram. D, E, Immunoblots of N2A cell lysates demonstrated that both 4.1N as well as IP6K2 translocate into the nucleus of N2A cells in the presence of NGF. However, in the absence of IP6K2, 4.1N failed to transfer into the nucleus of N2A cells. F, Experimental evidence that IP6K2 is a prime facilitator for nuclear translocation of 4.1N along with NGF in N2A cells. G, Immunoblots establish that NLS of IP6K2 is essential for the translocation of 4.1N into the nucleus, as 4.1N fails to translocate into the nucleus when N2A cells are transfected with IP6K2 lacking NLS. H, Double-immunofluorescence staining of N2A cells against IP6K2 (green) and 4.1N (red) reveals significant nuclear translocation of 4.1N in the presence of NGF and no nuclear expression of 4.1N in absence of IP6K2. Scale bar, 10 μm. Data are representative of three independent experiments performed under identical conditions. Data are presented as the mean ± SD: ***p < 0.001, **p < 0.01, *p < 0.05, analyzed by one-way ANOVA.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Translocation Assay, Western Blot, Expressing, Transfection, Double Immunofluorescence Staining

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: Rab 5 and SNX-27 form a ternary complex with IP6K2–4.1N. A, Workflow for screening proteins that bind to the IP6K2–4.1N complex. B, Immunoprecipitates (IPs) of Rab 5 proteins blotted with IP6K2 and 4.1N antibodies, respectively, revealed the binding of Rab 5 to both IP6K2 and 4.1N in wild-type mouse brain lysates. C, Immunoblot (IB) showed that SNX-27 binds to 4.1N and not to IP6K2. D, Western blot of wild-type and IP6K2-KO mice brain lysates showed reduced the protein expression of Rab5 and SNX-27 in IP6K2-KO mouse brain lysates. E, Quantitation of the relative expression of Rab 5 and SNX-27 depicted in D through normalization against the relative expression of the relevant input. F, Western blots depict reduced expression of some prominent Immediate Early Gene (IEG)-encoded proteins like Arc, Fos B, NGF, and Synaptophysin (Syn) in the IP6K2-KO mice brain lysates. G, Quantitation of the depicted. Western blots in F through normalization against the input is shown in the histogram. Data were statistically analyzed by paired Student's t test. Significant differences (*p < 0.05) were observed in comparison with WT controls. Data are presented as the mean ± SD and are representative of three independent experiments done under identical conditions.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Western Blot, Expressing, Quantitation Assay

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: Lack of IP6K2 and 4.1N, highly expressed granule cells proteins, leads to diminished proliferation of cerebellar cells. A, Immunofluorescent staining of wild-type mouse brain cerebellar sections showed localization of IP6K2 and 4.1N in granule cells of the cerebellum. B, Western blots for calbindin and calretenin, respective markers for Purkinje and granule cells, showed reduced expression of both proteins in mouse brain lysates lacking IP6K2. C, Histological comparison through H&E staining of WT and IP6K2 KO brain (IP6K2-KO; 8-week-old) sections showed substantial decreases in the molecular layer compared with the WT. GCL, Granule cell layer; PCL, Purkinje cell layer; ML, molecular layer. D, H&E-stained sections of IP6K2-KO mouse also showed substantially fewer granule cells in the cerebellar region of mutant mice compared with WT. E, F, Mice injected with EdU showed fewer proliferating cells (marked with white arrow heads) in IP6K2-KO mice compared with the WT. G, XTT assay performed with knockdown of K2 as well as K2 lacking binding site for 4.1N (202–261) in N2A cells also revealed lower cell viability compared with control shRNA. Scale bar, 50 μm. The data are presented as the mean ± SD: ***p < 0.001, **p < 0.01, analyzed by one-way ANOVA. The H&E staining of wild type (WT) and IP6K2-Knockout (KO) sagittal brain sections (A), Hippocampus (B) and Cerebellum (C) is shown in Figure 4-1.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Staining, Western Blot, Expressing, Mutagenesis, Injection, XTT Assay, Binding Assay, shRNA, Knock-Out

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: IP6K2 and 4.1N expressed in the granule cells of cerebellum affect Purkinje cell morphology and parallel fiber–Purkinje cell synapses. A, Purkinje cell volume, spine number, and spine density were significantly decreased in IP6K2-KO mice when compared with their WT counterparts. (top, 60× magnification; bottom, 100× magnification). B, Immunostaining of VGlut1/calbindin and VGlut2/calbindin in cerebellar Purkinje cells from WT and IP6K2-KO mice showed that the density of VGlut1 was markedly reduced in IP6K2-KO mice, indicating that knock-out mouse brain sections contained fewer parallel fiber–Purkinje cell synapses compared with WT mouse brain. However, there was no difference in the climbing fiber–Purkinje cell synapses of IP6K2-KO mouse cerebellum compared with wild type. Scale bar, 20 μm. C, Electron microscopic analysis of the cerebellar molecular layer from IP6K2-KO mouse brain (8 weeks old) also revealed fewer synapses compared with WT mouse brain. However, the ultrastructure of synapses in the cerebellum was relatively normal in the IP6K2-KO. Scale bar, 500 nm. Data were statistically analyzed by paired Student's t test. Significant differences (***p < 0.001, **p < 0.01) were observed in comparison with wild-type controls. Data are presented as the mean ± SD and are representative of three independent experiments performed under identical conditions. Golgi staining of IP6K2-knockout (KO) mice cerebellum with no significant morphological changes in pyramidal cells, granule cells, stellate cells and Golgi cells when compared to their wild-type (WT) counterparts, as shown in Figure 5-1.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Immunostaining, Knock-Out, Staining

Journal: The Journal of Neuroscience

Article Title: Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N

doi: 10.1523/JNEUROSCI.1165-18.2018

Figure Lengend Snippet: IP6K2-KO mice show deficiencies in motor coordination and locomotor activity. A, Motor learning and coordination were compared between the IP6K2-KO and WT mice by the rotarod test. This test revealed reduced motor learning and coordination in the IP6K2-KO mice compared with WT mice. B, General locomotor activity in IP6K2-KO mice was assessed by the open field analysis. Such behavioral analysis also revealed reduced peripheral as well as central activity in IP6K2-KO mice when compared with WT mice. C, Gait analysis in IP6K2-KO mice also showed significant decreases in stride length and average speed of movement with respect to WT mice. However, there was no significant change between the paw print width of the WT and K2-KO mice. Data shown are representative of three independent experiments done under identical conditions with the mean ± SD: ***p < 0.001, **p < 0.01.

Article Snippet: IP6K2 shRNA, 4.1N, and control shRNA plasmids were from Santa Cruz Biotechnology.

Techniques: Activity Assay